Combination antiretroviral drug regimens have proven remarkably successful at controlling HIV infection and have resulted in significant reductions in morbidity and mortality from AIDS in North America and Western Europe. Despite this success, new targets for inhibition of HIV replication are highly desirable, and new drugs will be needed to combat the rising problem of antiretroviral drug resistance. The overall goal of this research plan is to develop assays suitable for high throughput screening for the identification of small molecules that inhibit the HIV assembly process. The identification of specific HIV assembly inhibitors will serve two major purposes. First, these compounds will provide valuable tools for dissecting distinct stages of the assembly process at the cellular and molecular level. Second, some of the identified compounds may serve as lead compounds for the development of novel antiretroviral drugs. Here we describe an integrated research program designed to develop high-throughput screening assays for inhibitors of HIV assembly. Experiments in Specific Aim 1 will develop and validate a cell-based screening assay for inhibitors of Gag- Gag interactions using Gag-CFP:Gag-YFP FRET as the signal. This assay will be adapted to HTS capability, and automated screening of a compound library will be performed. In Aim 2, the FRET-based assay will be enhanced to produce two second-generation assays with improved performance using the latest fluorescent protein derivatives and assay technologies. A novel fluorescence anisotropy assay for measuring FRET will be evaluated. Experiments in Aim 3 will develop and validate an in-vitro screening assay for inhibitors of a new interaction discovered in our laboratory between Gag and the AP-3 complex. Cy3-Cy5 FRET will be the basis for the readout from this assay. Together, these assays should identify small molecule compounds that will inform the field of HIV assembly and provide important tools for further dissection of assembly pathways. These HTS assays can also be applied to screening of a number of libraries available through the Molecular Libraries Initiative, and are designed to address the goals of PA-04- 068, Development of Assays for High-Throughput Drug Screening.